The selective growth and generation of biological tissue is an area of biomedical scientific and technological advancement with a variety of useful applications. For example, selectively grown or generated biological tissue can be implanted into living beings to repair or replace biological tissue that has been damaged or lost due to injury or illness. Connective tissue, muscle tissue, nervous tissue, and epithelial tissue compose the organs and body structures of living beings, and the ability to selectively generate any or all of these tissue types for implantation and repair or replacement is desirable. However, it is extremely difficult to generate any tissue which perfectly mimics the biological and mechanical characteristics of the target tissue and to generate a tissue which the body will not reject as foreign upon implantation.
One method for selectively generating biological tissue to be implanted in a living being includes generating a portion of donor tissue that is similar or nearly identical to the tissue to be repaired and/or replaced, also referred to herein as “native” tissue. In some cases, the implanted tissue is nearly identical to the native tissue. In other cases, however, the implanted tissue is a precursor to the native tissue, and the implanted tissue serves as a scaffold for the further generation and integration of the native tissue within the body. In these latter cases, the implanted tissue must enable and facilitate growth and integration into existing tissue in the body. In either case, the implant tissue must enable and facilitate integration into the body and prevent rejection.
Generating tissue for implantation often includes decellularization of donor tissue to remove cellular and genetic material from the tissue. Decellularization can be achieved, for example, by applying a chemical decellularizing agent to the tissue. Decellularized tissue is beneficial because it contains the mature, healthy extracellular matrix (ECM) of the target tissue being repaired or replaced. Thus, native decellularized ECM is able to direct host cell infiltration, migration, and phenotype induction to promote integration of the implant tissue with the target tissue. Furthermore, the biological makeup of ECM components of various decellularized tissues is largely conserved and largely non-immunogenic. Thus, autologous, allogenic, and xenogenic transplantation of decellularized tissues with relatively minimized risk to the host is possible.
The efficacy of a decellularizing agent in producing acceptable decellularized tissue depends, in part, on the physical properties of the tissue being decellularized. For example, cartilage, and in particular articular cartilage, is generally composed of thick and dense tissue. Accordingly, the infiltration and/or penetration of decellularizing agents into the tissue, and thus the production of acceptable decellularized tissue, is inhibited by the structure of the tissue. Furthermore, after decellularization, the high tissue density of articular cartilage prevents cellular infiltration and remodeling in the long term, thus inhibiting integration of the implanted tissue. However, despite these challenges, generation and implantation of articular cartilage tissue is highly desirable, because articular cartilage is resilient to natural repair and is subject to high mechanical forces, which promotes further tissue degeneration.
Accordingly, methods have been developed to address the reduced cellular infiltration and remodeling of implant articular cartilage tissue. For example, in some methods, the ECM of the decellularized articular cartilage tissue is modified by guanidine-hydrochloride reduction of glycosaminoglycans (GAGs) to increase tissue porosity. However, such modifications to the ECM are often unsuccessful in promoting bulk cellular infiltration, which can lead to global degeneration and limited utility of the implant in the body.
Thus, further methods have been developed to help retain the local mechanical, structural, and biochemical microenvironment of native articular cartilage, but allow for easier penetration of decellularization agents as well as means to increase cellular infiltration into dense native tissue. For example, in some methods, atomized (e.g. pulverized or microparticulated) cartilage tissue is size controlled to create a powder of fragments of cartilage. Fragments are then either crosslinked to each other or suspended in a polymerizable medium to create three-dimensional constructs of tunable size and/or cartilage microparticle density, which can promote cell attachment and upregulation of cartilage specific genes (e.g. type II collagen, SOX9, aggrecan) to form neocartilage.
However, one problem with these methods is that crosslinking the fragments often requires conditions which are hostile to the tissue. Thus, cell-seeding, to add necessary cellular material back to the tissue, may be required after crosslinking. Cell-seeding is generally an imprecise and inefficient process, and usually results in cells that are not globally distributed in the three-dimensional construct. An additional problem with these methods is that they result in cartilage microparticle density that is largely dependent on the packing efficiency of the particles, but is otherwise uncontrolled. Furthermore, these methods do not facilitate investigation of the contribution of physiochemical properties to chondrogenic induction and maintenance, which could provide insight and direction to further improve selective tissue generation and integration. Thus, there remains a need to (1) create a more tunable, controlled system of cartilage microparticle construct formation without the need for hostile crosslinking, and (2) further examine the contribution of the complex native cartilage microenvironment to the induction of chondrogenic differentiation.